Microbial stem cell technology

ABSTRACT

The present disclosure relates to microbial stem cell technology that enables a growing microbial culture to stably maintain two or more distinct cell types in a ratio that can be genetically programmed and/or dynamically controlled during cultivation. It is contemplated that embodiments described herein can be utilized to increase product yield in microbial fermentations and advanced engineering of biomaterials using genetically engineered microbial cells, among others.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 16/393,671, filed Apr. 24, 2019 (now U.S. Pat. No. 11,525,117) which claims priority to U.S. Provisional Application Ser. No. 62/712,857, filed Jul. 31, 2018, and U.S. Provisional Application Ser. No. 62/661,818, filed Apr. 24, 2018, which are herein incorporated by reference in their entirety.

BACKGROUND Field

Embodiments of the present disclosure generally relate to microbial stem cell technology.

Description of the Related Art

Fermentation-based chemical production processes utilize batch microbial cultures to produce a variety of valuable biosynthetic products ranging from pharmaceuticals to biofuels. Such bioprocesses provide an alternative to traditional extraction and chemical production methods. However, a significant limitation is that current bioprocess technology is often not economically competitive with more traditional production methods. The relatively high cost of production using bioprocess technology comes from a number of sources, including high maintenance costs of bioreactor equipment, expensive reagents for inducing microbial production and cell growth, and low product production due to the toxic effects of bioproduction on the microbial cell culture. This toxicity, or biosynthetic burden, places significant limits on the maximum level of product synthesis and hinders the rate at which product-synthesizing bacterial cells can produce product and divide.

One common obstacle in achieving high biosynthetic product yield from bioprocesses is the rapid accumulation in such microbial cultures of genetic mutants where product synthesis is reduced or non-existent. Such mutants have a selective advantage over producing cells due to a lower metabolic burden or lower product toxicity experienced by the mutant when compared to the producing cells. As a consequence, a large fraction of the microbial culture can be overtaken by non-producing mutant cells, thus reducing overall product yield. Conventional methods utilized to restore product yield include renewing the microbial culture. However, culture renewal is time consuming, inefficient and increases technological complexity of the bioprocess.

Microbial cells used for some bioprocesses require special induction conditions, such as temperature or culture density, or specific chemical signals, such as small molecule inducing agents, in order to begin producing product. Using these techniques to achieve culture induction in large-scale bioprocess facilities can add significant expense.

Bioprocesses suffer another drawback when utilized to produce complex molecules. Complex molecules typically require a multi-step synthesis process in which multiple bacterial cultures are mixed to produce different intermediates of the molecule synthesis pathway. However, mixed cultures are very difficult to control and utilizing separate bioreactors for individual bacterial cultures is costly and inefficient.

Accordingly, what is needed in the art are improved methods and materials for controlling microbial cell cultures.

SUMMARY

In one embodiment, a method of establishing microbial cell types is provided. The method includes modifying microbial cells with a genetic circuit, the genetic circuit configured to produce a localization factor exhibiting an asymmetric localization pattern as a basis for asymmetric cell division, the asymmetric cell division facilitating establishment of distinct cell types within a population of microbial cells. The genetic circuit is also configured to produce a signaling factor linked to the localization factor to form a biochemical platform, the biochemical platform eliciting differentiable cell behavior in microbial cells that inherit the biochemical platform.

In another embodiment, a chemically inducible genetic for tuning a population distribution of microbial cells is provided. The chemically inducible genetic circuit includes a protein factor exhibiting subcellular polar localization for directing an establishment of distinct cell types within the population of microbial cells, an enzyme fused to the protein factor to form a complex, the enzyme establishing a gradient of a small molecule that elicits a programmable pattern of gene expression, and a chemically inducible promoter located upstream of genetic coding sequences of the complex.

In yet another embodiment, an optogenetic circuit for tuning a population distribution of microbial cells is provided. The optogenetic circuit includes a photo-controllable transcriptional regulation system, a localization factor exhibiting subcellular polar localization for directing an establishment of distinct cell types within a population of microbial cells, a signaling factor linked to the localization factor to form a biochemical platform, the signaling factor catalyzing production of a secondary messenger molecule that is asymmetrically distributed during cell division, the asymmetric distribution of the secondary messenger molecule facilitating a differential program of gene expression in two or more daughter cells, and a promoter linking the photo-programmable transcription regulation system to production of the localization factor and the signaling factor.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

So that the manner in which the above recited features of the present disclosure can be understood in detail, a more particular description of the disclosure, briefly summarized above, may be had by reference to embodiments, some of which are illustrated in the appended drawings. It is to be noted, however, that the appended drawings illustrate only exemplary embodiments and are therefore not to be considered limiting of its scope, and may admit to other equally effective embodiments.

FIG. 1A illustrates a conceptual diagram of a method for utilizing asymmetric cell division to generate distinct cell types according to an embodiment described herein.

FIG. 1B illustrates images of two cell types with a fluorescent signal overlaid on the image according to an embodiment described herein.

FIG. 1C illustrates a schematic diagram of a genetic circuit for controlling c-di-GMP levels in asymmetrically dividing cells according to an embodiment described herein.

FIG. 1D illustrates images of cell motility in agar of three cell types according to an embodiment described herein.

FIG. 1E illustrates images of cell motility in agar of three cell types according to an embodiment described herein.

FIG. 2A illustrates a conceptual diagram of a tripartite split-GFP reporter system according to an embodiment described herein.

FIG. 2B illustrates a schematic diagram of a genetic circuit for detecting c-di-GMP levels in asymmetrically dividing cells according to an embodiment described herein.

FIG. 2C illustrates a conceptual diagram of a method for utilizing the split-protein c-di-GMP reporter system to detect c-di-GMP levels in asymmetrically dividing cells according to an embodiment described herein.

FIG. 2D illustrates images of two cell types cultured on plated media at an indicated time according to an embodiment described herein.

FIG. 2E illustrates images of cells at indicated times during cellular division and corresponding flow cytometry analysis of the cell populations according to an embodiment described herein.

FIG. 2F illustrates images of cells at indicated times during cellular division and corresponding flow cytometry analysis of the cell populations according to an embodiment described herein.

FIG. 3A illustrates a schematic diagram of a genetic circuit according to an embodiment described herein.

FIG. 3B illustrates images of cells at indicated times during cellular division and corresponding flow cytometry analysis of the cell populations according to an embodiment described herein.

FIG. 3C illustrates images of cells at indicated times during cellular division.

FIG. 3D illustrates flow cytometry analysis of the cell populations of FIG. 3B under modified induction conditions.

FIG. 3E illustrates flow cytometry analysis of the cell populations of FIG. 3B under modified induction conditions.

FIG. 3F illustrates flow cytometry analysis of cell populations during cellular division according to an embodiment described herein.

FIG. 3G illustrates flow cytometry analysis of cell populations during cellular division according to an embodiment described herein.

FIG. 3H illustrates flow cytometry analysis of cell populations during cellular division according to an embodiment described herein.

FIG. 3I is a graph illustrating GFP intensities of PopZ-negative cells from FIGS. 3A-3H at the 4 hour time point according to an embodiment described herein.

FIG. 3J is a graph illustrating the average intensity of mChy fluorescence and average number of mChy-positive cells at indicated times during cellular division according to an embodiment described herein.

FIG. 3K is a graph illustrating the average number of YhjH-mChy-PopZ foci per cell at indicated times during cellular division according to an embodiment described herein.

FIG. 3L illustrates images of cells expressing YhjH-mChy-PopZ, followed by induction of asymmetric cell division associated with the expression of waxy ether biosynthesis in one of the cell types according to an embodiment described herein.

FIG. 4A illustrates a schematic diagram of a genetic circuit according to an embodiment described herein.

FIG. 4B illustrates images of cells at indicated times during cellular division with changing light conditions according to an embodiment described herein.

FIG. 4C is a graph illustrating the fraction of PopZ-positive cells at the indicated time points from FIG. 4B according to an embodiment described herein.

FIG. 4D illustrates images of cell motility in agar under different light regimes according to an embodiment described herein.

FIG. 4E illustrates images of cell motility in liquid suspension under different light regimes according to an embodiment described herein.

FIG. 5A illustrates a physical map of a plasmid listed in Table 1 according to an embodiment described herein.

FIG. 5B illustrates a physical map of a plasmid listed in Table 1 according to an embodiment described herein.

FIG. 5C illustrates a physical map of a plasmid listed in Table 1 according to an embodiment described herein.

FIG. 5D illustrates a physical map of a plasmid listed in Table 1 according to an embodiment described herein.

FIG. 5E illustrates a physical map of a plasmid listed in Table 1 according to an embodiment described herein.

FIG. 5F illustrates a physical map of a plasmid listed in Table 1 according to an embodiment described herein.

FIG. 5G illustrates a physical map of a plasmid listed in Table 1 according to an embodiment described herein.

FIG. 5H illustrates a physical map of a plasmid listed in Table 1 according to an embodiment described herein.

FIG. 5I illustrates a physical map of a plasmid listed in Table 1 according to an embodiment described herein.

FIG. 5J illustrates a physical map of a plasmid listed in Table 1 according to an embodiment described herein.

FIG. 5K illustrates a physical map of a plasmid listed in Table 1 according to an embodiment described herein.

FIG. 5L illustrates a physical map of a plasmid listed in Table 1 according to an embodiment described herein.

FIG. 5M illustrates a physical map of a plasmid listed in Table 1 according to an embodiment described herein.

FIG. 5N illustrates a physical map of a plasmid listed in Table 1 according to an embodiment described herein.

FIG. 5O illustrates a physical map of a plasmid listed in Table 1 according to an embodiment described herein.

FIG. 5P illustrates a physical map of a plasmid listed in Table 1 according to an embodiment described herein.

FIG. 5Q illustrates a physical map of a plasmid listed in Table 1 according to an embodiment described herein.

Figures included herein illustrate various embodiments of the disclosure. It is contemplated that elements and features of one embodiment may be beneficially incorporated in other embodiments without further recitation.

DETAILED DESCRIPTION

The present disclosure relates to microbial stem cell technology that enables a growing microbial culture to stably maintain two or more distinct cell types in a ratio that can be genetically programmed and/or dynamically controlled during cultivation. It is contemplated that embodiments described herein can be utilized to increase product yield in microbial fermentations and advanced engineering of biomaterials using genetically engineered microbial cells, among others.

Asymmetric bacterial cell division produces daughter cells with different identities and patterns of gene expression. Polarized distributions of regulatory proteins and the associated asymmetry in signaling networks within bacterial cells often influence such division and gene expression. Cellular division and gene expression are fundamental to multicellular development and the benefits that are derived from collaborating cell types.

In one embodiment, a method for inducing a program of asymmetric cell division coupled with cell differentiation in microbial cells is provided. The method includes modifying microbial cells by placing in them a genetic circuit that is configured to produce an asymmetrically localized biomolecular platform. This biomolecular platform, in addition to having the property of asymmetric localization in pre-divisional cells, also has the function of driving a definable pattern of cell differentiation during cell division. A consequence is the establishment of multiple distinct, differentially controllable cell types within the population of microbial cells.

In one embodiment, a chemically inducible biochemical platform for establishing programmable asymmetric cell division in microbial cells is provided. The biochemical platform includes a protein that localizes to a discrete location or set of defined locations in the cell, and subsequently becomes asymmetrically distributed between daughter cells in the process of cell division. Asymmetric localization of this biochemical platform, including but not limited to the vicinity of the cell poles, serves as the basis for directing differentiation between daughter cells during cell division. In addition to having a localization activity, the biochemical platform also includes a directly or indirectly linked signaling factor which establishes a biologically differentiable trait that distinguishes those daughter cells that inherit the platform from siblings that do not.

In one embodiment, an optogenetic circuit for tuning a population distribution of microbial cells based on the expression or activity of the biochemical platform described above is provided. The optogenetic circuit includes a light-controllable transcriptional regulation system, which controls the expression or activity of said biochemical platform.

In one embodiment described herein, cell geometry and transcriptional control elements from multiple bacterial species are combined to create a unique and robust synthetic genetic circuit for establishing programmable asymmetric cell division in commercially significant bacterial species such as E. coli. For example, individual components include the polar organizing protein PopZ from Alphaproteobacteria and regulators of c-di-GMP dependent transcriptional activity from Klebsiella and other organisms.

According to embodiments described herein, seemingly complex biological phenomenon—asymmetric cell division and the generation of differentiated cell types—can be brought about by a small set of genes in a prokaryotic organism. The transcriptional output of the genetic circuit is directed to establish different sub-populations of differentiated cells. For example, the transcriptional output of the genetic circuit may establish motile versus non-motile cells or biosynthetically productive cells versus non-productive cells. In the productive cell versus non-productive cell implementation, productive “factory” cells express a set of enzymes in a biosynthetic pathway, while non-productive cells function as a regenerative population of stem cells, which produce a factory and a non-factory cell with every cell division. Thus, a first sub-population of differentiated cells established by methods herein may function to produce a biosynthetic product while a second sub-population functions to maintain the two or more sub-populations. Differentiable cell behavior traits may also include, for example, distinct programs of gene expression or differences in protein complex assembly.

In one embodiment described herein, population distribution of multiple cell types can be tuned using a chemically-regulated system that regulates expression of the localization factor by exposure to one or more chemicals. For example, population distribution of cell types may be tuned using a chemical inducer and a chemically-inducible promoter located upstream of genetic coding sequences of the localization factor. Alternatively, population distribution of cell types may be tuned using a chemical repressor.

Embodiments described herein also provide for population distribution of cell types which can be tuned using a light-activated system that regulates expression of the localization factor by exposure to certain wavelengths of light. In one embodiment, population distribution of cell types can be tuned using a light-activated system that regulates expression of the localization factor by exposure to light having a wavelength in the visible, infrared (IR), and/or ultraviolet (UV) portions of the electromagnetic spectrum. For example, population distribution of cell types can be tuned by exposure to red or green light. In some embodiments, the light-activated system regulates expression of the localization factor by controlling the activity of one or more signal transduction proteins. In one embodiment, the light-activated system regulates expression of the localization factor by controlling the activity of a histidine kinase.

The synthetic genetic circuits described herein provide a genetically programmable platform for leveraging asymmetric cell division of a genetically uniform population of microbial cells to create new types of collaborative microbial communities. More specifically, microbial stem cell technology enables maintenance of “stem” cells which are not burdened with biosynthetic product synthesis. Upon cell division, a “stem” cell produces daughter “factory” cells that are genetically programmed for product synthesis. The lack of selective pressure maintains “stem” cells free of mutations and capable of continually generating product synthesizing “factory” cells. In one embodiment, a ratio of “stem” to “factory” cells is genetically programmed and/or controlled by physiochemical stimuli, such as exposure to chemicals, light, and/or changes in temperature.

In order to produce different cell types, such as “stem” and “factory” cell types, a localization factor that accumulates at an asymmetric location in a pre-divisional cell is utilized. In one embodiment, the localization factor is a protein factor. The localization factor may be any suitable protein that self-assembles into a large complex or macromolecular structure, and/or accumulates in a certain subcellular locations. In one example, the localization factor accumulates at one or both cell poles in various rod-shaped bacteria. In one embodiment, the localization factor is a polar organizing protein PopZ, or a homolog thereof. PopZ is a conserved protein from the class Alphaproteobacteria. In E. coli, a common host of bacterial fermentations, PopZ compactly accumulates at a single cell pole. When such a cell divides, the cell produces one daughter cell with PopZ (the “stem” or “progenitor” cell) and another daughter cell without PopZ (the “factory” cell), illustrated in the conceptual diagram of FIG. 1A.

In the absence of the polar PopZ, the “factory” cell continues to divide and give rise to new “factory” cells while the “stem” cell retains PopZ over several hours. With every cell division of the “stem” cell, the “stem” cell will continue to produce one new “factory” cell. In one embodiment, PopZ is fused to a signaling factor that establishes a gradient of a small molecule that facilitates the asymmetric distribution of PopZ protein in a programmable pattern of gene expression. The signaling factor may positively or negatively affect the gene expression of the small molecule. For example, the signaling factor may be an enzyme that catalyzes the production of a small molecule. The signaling factor may further be directly or indirectly linked to the PopZ protein. In one embodiment, the small molecule is a secondary messenger molecule. In one embodiment, the small molecule is a cyclic nucleotide-based second messenger. For example, the small molecule is cyclic-diguanosine monophosphate (c-di-GMP), cyclic-adenosine-monophosphate (cAMP), cyclic-di-adenosine monophosphate (c-di-AMP), cyclic guanosine monophosphate (cGMP), and c-di-AMP/GMP.

The PopZ protein may be fused to any suitable signaling factor to form a PopZ-signaling factor complex establishing a gradient of the small molecule regulating gene expression. For example, the PopZ protein may be fused to an enzyme, such as a kinase, phosphatase, protease, protease adaptor protein, or the like. In one embodiment, PopZ is fused to an enzyme that produces c-di-GMP or a precursor thereof. In one embodiment, PopZ is fused to an enzyme that specifically degrades c-di-GMP. For example, PopZ is fused to a phosphodiesterase. Thus, cells that retain PopZ at a single pole also maintain a low level of intracellular c-di-GMP, whereas “factory” cells have higher levels of naturally synthesized intracellular c-di-GMP.

A genetic element may be further utilized to induce the expression of enzymes that catalyze synthesis a desired biosynthetic product. In one embodiment, the enzyme is diguanylate cyclase and synthesizes c-di-GMP from the intracellular pool of guanosine triphosphate (GTP). In one embodiment, the genetic element includes a c-di-GMP-sensitive transcription factor MrkH and a PmrkA promoter regulated by MrkH. It is believed that the genetic element can be utilized to differentially control gene expression of a desired biosynthetic product. For example, PmrkA is utilized to drive expression of a gene encoding a desired fermentation product(s). As c-di-GMP accumulates in “factory” (but not “stem”) cells, the c-di-GMP binds to MrkH, which in turn binds to the PmrkA promoter and activates target gene expression. Thus, asymmetric cell division may be used to induce production in cells that lacking the localization factor.

In one embodiment, genetic engineering utilizes a factor, such as the localization factor described above, that exhibits properties of subcellular localization as the basis for directing the establishment of distinct cell types in a population of microbial cells.

In another embodiment, the factor is utilized as the basis for controlling gene expression of a multi-gene biosynthetic product pathway.

In another embodiment, a group of genetic cassettes that utilize the factor that has the property of subcellular localization are utilized as the basis for directing the establishment of distinct cell types in a population of microbial cells.

In another embodiment, a group of genetic cassettes carry the sequences encoding the PopZ-signaling factor complex utilized as the basis for directing the establishment of distinct cell types in a population of microbial cells.

In another embodiment, the PopZ-signaling factor complex is further fused to a third protein to form a tripartite protein complex utilized as the basis for directing the establishment of distinct cell types in a population of microbial cells.

In one embodiment, the signaling factor is a member of a split protein system, and is utilized as the basis for conditional reconstitution of protein activity.

In another embodiment, Caulobacter crescentus PopZ, a homolog of PopZ, or a variant or fragment thereof, is utilized as the basis for directing the establishment of distinct cell types in a population of microbial cells.

In another embodiment, a bacterial polar organizing protein, or a variant or fragment thereof, is utilized as the basis for directing the establishment of distinct cell types in a population of microbial cells.

In another embodiment, a polar landmark or hub protein, such as HubP from the gammaproteobacteria class of bacteria, is utilized as the basis for directing the establishment of distinct cell types in a population of microbial cells.

In another embodiment, an outer membrane autotransporter protein, such as IcsA, is utilized as the basis for directing the establishment of distinct cell types in a population of microbial cells.

In another embodiment, multiple cell types with distinct patterns of gene expression from a genetically uniform population of microbial cells are established and controlled by the use of a factor that has the following properties: subcellular localization, and the ability to control gene expression, either through direct manipulation of transcriptional machinery or indirectly through control of factors that regulate transcriptional activity.

In another embodiment, different patterns of gene expression among distinct cell types in a population of microbial cells are controlled through a mechanism that regulates the levels of a secondary messenger, such as a small molecule signal.

In another embodiment, different patterns of gene expression among distinct cell types in a population of microbial cells are controlled through a mechanism that uses a factor that has subcellular localization characteristics as the basis for controlling differential gene expression.

In another embodiment, protein-protein interactions in a population of microbial cells can be controlled through a mechanism that uses a factor that has subcellular localization characteristics. For example, the mechanism may be utilized to conditionally reconstitute a split protein system, such as green fluorescent protein (GFP).

In one embodiment, the signaling factor is a member of a split protein system.

In another embodiment, the system is not limited to E. coli but may work in many other cell types (prokaryotic and eukaryotic).

In another embodiment, the system components (geometric organizer, secondary messengers, transcription control, etc.) can originate from any organism. In one embodiment, components from Caulobacter, Klebsiella, Synechocystis, Rhodobacter, Xanthomonas, and plasm ids of various types are utilized.

In another embodiment, various second messengers (in addition to c-diGMP) can be used to establish control of gene regulation, both nucleotide messengers and non-nucleotide. For example, cyclic adenosine monophosphate (cAMP) or calcium ions may be utilized to establish control.

In another embodiment, the output of the system does not have to be transcriptional activity, but could be other types of activity, such as motor protein activity, enzymatic activity or protein interaction.

In one embodiment, the genetic circuit is used to separate a multi-step biosynthetic pathway into multiple stages, each stage being activated in a distinct differentiated cell type within a community of microbial cells. For example, distinct components or precursor of a desired biosynthetic product may be produced in distinct cell types.

In another embodiment, the localization factor may be non-localized to the cell pole, and it may be non-homologous to PopZ.

In another embodiment, multiple PopZ or other geometric organizing proteins can be applied to diversify the activities that are being controlled.

In another embodiment, various stimuli can control the system, including a broad range of electromagnetic wavelengths, chemical, thermal, or mechanical stimuli. For example, small molecules and visible light are suitable for utilization as independently usable stimuli.

Experimental Methods and Materials Formation of Different Cell Types Based on Differential Accumulation of Small Molecules

Table 1 below provides a summary of plasmids utilized in the experimental methods and procedures described herein. It is contemplated that any suitable vector may be utilized to carry out the methods described herein.

TABLE 1 Anti- biotic Plasmid resis- name Essential genes and regulatory elements tance pBAD araBADp promoter (SEQ ID No. 2); MCS Amp pACYC T7p promoter (SEQ ID No. 22); MCS Cam pBAD-YmP yhjH-mCherry-popZ fusion protein (under Amp araBADp promoter) (SEQ ID No. 28) pBAD-YmP-B yhjH-mCherry-popZ (under araBADp Amp promoter); bphS-bphO (under tetp promoter) (SEQ ID Nos. 3-4) pBAD-YmP-S yhjH-mCherry-popZ (under araBADp Amp promoter); slr1143 (under tetp promoter) (SEQ ID No. 20) pAC-YmP-B yhjH-mCherry-popZ (under T7p promoter); Cam bphS-bphO (under tetp promoter) pAC-YmP-S yhjH-mCherry-popZ (under T7p promoter); Cam slr1143 (under tetp promoter) pMQ-132 pilZ-gfp10 (SEQ ID No. 11); fimX-gfp11 Gent split GFP (under lacp promoter) (SEQ ID No. 9); gfp1-9 (under T7p promoter) (SEQ ID No. 10) pMAL-Slr1143 slr1143 (under tacp promoter) Amp pMAL-p2x tacp promoter; MCS Amp pBAD-MrkH c-di-GMP dependent transcription factor Amp mrkH (SEQ ID No. 15) under araBADp promoter pB-Mrk-GFP mrkH (under araBADp promoter); monomeric Amp superfolder msf-gfp (under mrkAp promoter) (SEQ ID No. 16) pB-Mrk-rbsGFP mrkH (under araBADp-promoter); msf-gfp Amp (under mrkAp promoter with strong RBS) pCDF-pMrkA- mrkAp-gfp with restriction sites downstream Spec GFP(−) msf-gfp for insertion of additional components pBAD-M-G-W mrkH (under araBADp-promoter); gfp, ac-CoA Amp reductase & WE synthase as a poly-cistronic message under mrkAp promoter pSR58-6 ccaR response regulator (SEQ ID No. 6); Cam (pCR) PcpcG2-172 promoter, controlling expression of GFP pCRP ccaR response regulator; ccaRp promoter Cam (SEQ ID No. 7), yhjH-mCherry-popZ pNO286-3 ccaS light inducible histidine kinase (SEQ ID Spec (pCS) No. 8); ho1 & pcyA genes (SEQ ID Nos. 12, 17), responsible for PCB chromophore biosynthesis pCRPMG ccaR response regulator; ccaRp promoter; Cam yhjH-mCherry-popZ; mrkH (under araBADp promoter); mrkAp promoter; msf-gfp reporter pCSTS ccaS light inducible histidine kinase; ho1 & Spec pcyA genes, responsible for PCB chromophore biosynthesis; slr1143 (under tetp promoter control) pCRPMG-Mot ccaR response regulator; ccaRp promoter; Cam yhjH-mCherry-popZ; mrkH (under araBADp promoter); mrkAp promoter, controlling expression of msfGFP and motA, motB genes

FIG. 1A illustrates a conceptual diagram of a method for using polar asymmetry to generate two distinct cell types. “Induction” refers to the production of the biochemical platform, herein labeled “YhjH-mChy-PopZ,” having the localization factor PopZ. “Wash-out” refers to a stoppage in biochemical platform production. Asymmetric cell division occurs subsequent to “wash-out”. “High c-di-GMP” and “low c-di-GMP”, together with the “YhjH” and “Slr” signaling components of the genetic circuit, provide an example of a system that gives rise to differentiable cell types based on differentiable cell behavior.

To facilitate phenotypic differences between “stem” cells and “factory” cells, the PopZ protein was linked to the c-di-GMP signaling system by fusing PopZ (SEQ ID No. 18) with a c-di-GMP phosphodiesterase YhjH (SEQ ID No. 25) from E. coli and a red fluorescent protein mCherry (mChy) (SEQ ID No. 13). YhJH and mChy were translationally fused with the N-terminus of PopZ using 9- and 12-amino acid linker sequences to form the tripartite YhJh-mChy-PopZ biochemical control platform.

FIG. 1B includes time lapse fluorescence microphotographs depicting the asymmetric cell division generating of two distinct cell types characterized by the presence of the YhjH-mChy-PopZ biochemical platform (exhibiting red fluorescence) over a 200 minute time course. To confirm whether YhjH-mChy-PopZ forms polar complexes that asymmetrically distribute between dividing cells, the YhjH-mChy-PopZ biochemical platform was expressed in E. coli cells of the strain MG-1655 DE3. The cells were transformed with the plasmid pBad-YmP (plasmid map shown in FIG. 5A) having YhjH-mChy-PopZ fused downstream of an araBAD promoter via isothermal Gibson Assembly, thus having expression of the YhjH-mChy-PopZ biochemical platform regulated by arabinose. FIG. 1C illustrates the genetic circuit comprising the YhJh-mChy-PopZ biochemical platform, wherein YhjH catalyzes the hydrolysis of c-di-GMP into pGpG, a linear diguanylate and hydrolysis product of c-di-GMP, and guanosine monophosphate (GMP).

The MG-1655 DE3 cells were grown at 37° C. overnight in lysogeny broth (LB) media with the addition of appropriate antibiotics. The cells were then diluted 100 times and grown on a rotary drum in 2 ml volumes in glass tubes for 2 hours prior to protein expression activation. Expression of the YhjH-mChy-PopZ biochemical platform was induced for two hours using 0.2% of L-arabinose, followed by wash-out and removal of the L-arabinose. The L-arabinose inducer was removed by three repeats of pelleting the cells with micro-centrifugation at 9000 rpm and re-suspending the cells in fresh LB media. Prior to the subsequent chase period in which cells were inoculated without the inducer, the cells were diluted 5 times and incubated at 37° C. in 150 ml flasks with shaking at 250 rpm. The growing cultures of cells were continuously diluted by removing portions of media containing cells and adding fresh LB media to keep the cell cultures in a log growth phase, with an optical density (OD 600) in a range between 0.3 to 0.6.

To obtain the live-cell images in FIG. 1B, cells were immobilized on 1% agarose pads and imaged with a Zeiss Axio Imager Z2 epifluorescence microscope equipped with a Hamamatsu Orca-Flash4 sCMOS camera and a Plan-Apochromat 100×/1.46 Oil Ph3 objective. A Zeiss filter set 63HE was utilized to acquire the fluorescent signal of mChy and the images were collected and processed with Zen Blue software. It was observed that after transient expression of YhjH-mChy-PopZ, the next cell division was asymmetric with respect to the inheritance of PopZ and the accumulation of c-di-GMP levels.

To confirm whether the tripartite YhjH-mChy-PopZ biochemical platform retained phosphodiesterase activity upon transformation, YhJh-mChy-PopZ was expressed in an E. coli mutant strain MG1566 ΔyhjH with impaired motility in the presence of high c-di-GMP levels and compared to the wild-type MG1655 strain, which is characterized as being constitutively motile. The motility phenotype associated with the mutant MG1566 ΔyhjH strain in semi-solid agar was rescued by transforming the mutant cells with the plasmid pBad-YmP (plasmid map shown in FIG. 5A) and inducing expression of the tripartite YhJh-mChy-PopZ biochemical platform, thus suggesting that phosphodiesterase activity was retained. FIG. 1D illustrates swarm motility results of the mutant strain MG1566 ΔyhjH (left), the wild-type strain MG1655 (center), and the transformed mutant strain (right). To test the motility phenotype, 3 μl of log-phase cells were spotted on semi-solid agar plates comprising 1% tryptone, 0.5% sodium chloride, and 0.25% agar. The cells were grown for 7 hours at 37° C. in presence of 0.2% L-arabinose.

The difference in intracellular c-di-GMP levels between “factory” cells and “stem” cells was further enhanced by introduction of a c-di-GMP synthesizing enzyme diguanylate cyclase (DGC), depicted in the genetic circuit diagram in FIG. 1C. It is contemplated that modest DGC activity can raise c-di-GMP levels in “factory” cells while not overcoming the c-di-GMP hydrolytic activity of the YhjH-mChy-PopZ biochemical platform in “stem” cells. To determine the influence of DGC on the asymmetrically dividing cell lines, MG-1655 DE3 cells capable of gene expression using a T7 promoter expression platform were transformed with the plasmid pAC-YmP-B or pAC-YmP-S (plasmid maps shown in FIG. 5B-5E).

FIG. 1E illustrates the motility of these cells under conditions of DGC induction or no induction. The plasmid pAC-YmP-B (middle panels) included a less active DGC, BphS, while the plasmid pAC-YmP-S (lower panels) included a stronger DGC, Slr1143. The right panels of Figure S1B depict cells under strong DGC induction in the presence of 0.1 μg/ml anhydrotetracylcine, while the left panels depict basal DGC expression from the leaky tetp promoter. The cells transformed with pAC-YmP-S exhibited inhibited motility under both strong DGC induction and basal DGC expression, whereas cells transformed with pAC-YmP-B were induced with anhydrotetracycline to achieve inhibited motility. The control host strain, not bearing any plasmids, retained the motile state (FIG. 1E upper panel).

Next, the C-di-GMP levels in “factory” and “stem” cells were monitored using a fluorescent tripartite split-protein c-di-GMP reporter system having the Xanthomonas campestris proteins FimX and PilZ translationally fused to a green fluorescent protein (GFP) beta-barrel, as illustrated in FIG. 2A. FimX and PilZ were selected due to their enhanced interaction in the presence of c-di-GMP. Beta-strand 11 (GFP₁₁) of GFP was fused to the C-terminus of the c-di-GMP binding EAL domain of FimX and beta-strand 10 (GFP₁₀) of GFP was fused to the N-terminus of PilZ, respectively, using 10-amino acid long flexible GS-linkers. The remaining non-fluorescent portion of the GFP beta-barrel comprising beta-strands 1-9 (GFP₁₋₉) was expressed separately.

To clone the DNA constructs of the split-GFP c-di-GMP reporter system, nucleotide sequences encoding FimX (XccFimX^(EAL)), PilZ (XccPilZ₁₀₂₈), and split GFP fragments (GFP₁₀, GFP₁₁, and GFP₁₋₉) were codon-optimized and synthesized as two DNA strings. The final construct (SEQ ID No. 32) was assembled via Gibson assembly procedure, on a pMQ132 plasmid (plasmid map shown in FIG. 5F). FIG. 2B illustrates genetic circuits comprising the split-protein c-di-GMP reporter system.

It is contemplated that the FimX domain binds to the PilZ domain in the presence of c-di-GMP, thus bringing GFP₁₀ and GFP₁₁ together. The close proximity of GFP₁₀ and GFP₁₁ causes the spontaneous formation of an antiparallel beta sheet complementing GFP₁₋₉, resulting in GFP fluorescence. FIG. 2C illustrates a conceptual diagram of a method for using the split-protein c-di-GMP reporter system in combination with the YhjH-mChy-PopZ biochemical platform to monitor both “factory” cells and “stem” cells via characteristic c-di-GMP levels.

To characterize the interactions of Fim X and PilZ, fluorescence was compared between cells of the E. coli strain BL21 (DE3) expressing components of the split-GFP system and transformed with either the plasmid pMAL-Slr1143 having the highly active DGC Slr1143 or empty plasmid, as illustrated in FIG. 2D. Individual bacterial colonies were streaked on LB media plates supplemented with appropriate antibiotics and 10 μM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 37° C. for 24 hours. FIG. 2D illustrates the same plate under white light (left panel) and ultraviolet light, which excites GFP fluorescence (center panel). The relative levels of c-di-GMP levels in the cells were observed indirectly by Congo Red staining, which labels the curly fimbriae that are produced by E. coli in response to c-di-GMP (right panel). The split-GFP c-di-GMP reporter system produced fluorescence in high c-di-GMP cells expressing DGC Slr1143 (top sectors) and exhibited little or no fluorescence in low c-di-GMP cells lacking DGC Slr1143 (bottom sectors).

The split-GFP c-di-GMP reporter system was further tested using two genetic circuits differing in the level of DGC activity, also depicted in FIG. 2B (top right). Cells of the strain MG-1655 DE3 were transformed with either the pBAD-YmP-B or pBAD-YmP-S plasmid (plasmid maps shown in FIGS. 5B-5E) containing components for controlling c-di-GMP levels (YhjH-mChy-PopZ biochemical platform and a DGC) and the pMQ132-splitFRP plasmid (plasmid map shown in FIG. 5F) containing components for detecting c-di-GMP (split-protein c-di-GMP reporter system). Cells transformed with the pBAD-YmP-B plasmid expressed the less active DGC, BphS, while cells transformed with the pBAD-YmP-S plasmid expressed the more active DGC, Slr1143.

FIGS. 2E and 2F depict fluorescence microphotographs (top panels) and flow cytometry data (lower panels) of the cells over a 4 hour time course. After a two-hour induction period with 0.2% L-arabinose, 80-90% of the cells contained the YhJh-mChy-PopZ biochemical platform and exhibited little to no GFP fluorescence. After subsequent rounds of cell division in the absence of the inducer, the fraction of GFP-positive cells (“factory” cells) and the intensity of GFP signal increased while the fraction of cells containing YhjH-mChy-PopZ foci (“stem” cells) decreased. Furthermore, the percentage of double-positive cells was less than 1%. Thus, asymmetric division of the MG-1655 DE3 cells produced two cell types that can be differentiated on the basis of c-di-GMP levels.

Moreover, the cell types produced are functionally differentiable on the basis of the c-di-GMP dependent tripartite YhJh-mChy-PopZ. The flow cytometry data (lower panels) demonstrates that the main cell population experiences a transient dark phase, when they have neither YhjH-mChy-PopZ nor GFP expression. These cells may be low in c-di-GMP levels because they are only a few divisions away from the YhjH-mChy-PopZ containing ancestor, and have not yet accumulated sufficient c-di-GMP for assembly of the split-GFP c-di-GMP reporter system. Consistent with this interpretation, cells expressing the stronger of the two DGCs, Slr1143 (FIG. 2E), exhibited higher GFP levels and had a lower fraction of double negative cells compared to cells expressing BphS (FIG. 2F).

Fluorescence microphotographs were generated using the methods described above while also using a Zeiss Filter set 38HE to acquire GFP fluorescent signals and overlaying GFP and mChy signals on a phase contrast image (grayscale). For flow cytometry analysis, 200 μL samples of cells were fixed in 4% paraformaldehyde for 30 minutes, then washed and incubated in phosphate-buffered saline, before being stored at 4° C. From 100,000 to about 500,000 cells from each sample were analyzed by a Yeti Cell Analyzer flow cytometer (Propel Labs, Ft. Collins, Co.) utilizing Everest software. Cells were gated using linear forward scatter (FS) by log side scatter (SS), followed by gating on FS area by FS height for aggregate exclusion. Fluorescence data was collected using a 525/35 nm filter from the 488 nm laser line for GFP and a 615/20 nm filter from the 561 nm laser line for mChy. The data was analyzed using Kaluza Flow Cytometry Analysis Software (Beckman Coulter Life Sciences, Indianapolis, Ind.). For all flow cytometry data plots, fluorescence intensity of GFP is plotted on the X axis while mChy is plotted on the Y axis. The flow cytometry data plots in FIG. 2E show data from a single trial. All samples were collected, prepared, stored, and analyzed using the same methods.

Translating Differential Accumulation of Small Molecules into Differential Gene Expression

To facilitate differential gene expression patterns between “stem” cells and “factory” cells having asymmetric c-di-GMP distribution, a c-di-GMP-dependent transcriptional factor and reporter system from Klebsiella pneumoniae was utilized instead of the split-GFP c-di-GMP reporter system. The transcriptional factor and reporter system includes a transcriptional activator MrkH, which binds to a cognate promoter mrkAp (SEQ ID No. 14) in the presence of c-di-GMP, illustrated in the genetic circuit diagram of FIG. 3A (bottom). Binding of MrkH with mrkAp therein activates expression of downstream genes, such as gfp. The components for c-di-GMP dependent expression of GFP were inserted into a pBAD plasmid vector, resulting in a pBAD-Mrk-GFP plasmid providing moderate levels of GFP expression or a pBAD-Mrk-rbs-GFP plasmid providing higher levels of GFP expression (plasmid maps shown in FIGS. 5H-5I). To make the MrkH-mrkAP transcriptional factor and reporter system (SEQ ID No. 29) compatible with the tripartite YhJh-mChy-PopZ biochemical control platform, YhJh-mChy-PopZ expression was transferred to a pACYC plasmid backbone having an IPTG-inducible T7 promoter expression platform such as pAC-Ymp-B or pACYC-YmP-S (top) (plasmid maps shown in FIGS. 5B-5E).

To clone the MrkH-mrkAP transcriptional factor and reporter system into a pBAD plasmid, the coding sequence for MrkH, followed by a bi-directional terminator, was amplified from a template plasmid and cloned into the pBAD plasmid via isothermal Gibson assembly, resulting in a pBAD-MrkH plasmid (plasmid map shown in FIG. 5G). Subsequently, the mrkAp promoter sequence, and a GFP coding sequence were cloned downstream of the terminator and in the opposite transcriptional direction to mrkH, resulting in a pB-Mrk-GFP plasmid (plasmid map shown in FIG. 5H). As an alternative, MrkAp-gfp was modified by insertion of a stronger RBS upstream GFP. Isothermal assembly of mrkAp-rbs and gfp into the pBAD-MrkH plasmid vector produced the plasmid pB-Mrk-rbsGFP, providing a higher level of GFP translation.

MG-1655 DE3 cells were transformed with one of the pAC-Ymp-B or the pAC-YC-YmP-S plasmid and one of the pB-Mrk-GFP or the pBad-Mrk-rbs-GFP plasmid (plasmid maps shown in FIGS. 5B-5E, 5H and 5I) and then analyzed over a 4 hour time course following a 1.5 hour pulse of YhjH-mChy-PopZ expression induced with 0.02 mM IPTG. The same methods described above for fluorescence microscopy and flow cytometry were used for analysis. Similar to the GFP fluorescence levels observed with the split-GFP c-di-GMP reporter system in FIG. 2E, cells containing the YhJh-mChy-PopZ biochemical platform did not exhibit GFP expression while cells lacking the YhJh-mChy-PopZ biochemical platform exhibited high GFP expression.

FIGS. 3B-3E depict GFP expression in cells transformed with pAC-YmP-S and pB-Mrk-rbsGFP plasmids. The plasmid pBad-Mrk-rbs-GFP includes a highly active ribosome binding site (RBS) for mrkAp, RBS 1200, as compared to the natural RBS 300. The stronger RBS inserted upstream of GFP by isothermal assembly. It is contemplated that by expression of the activator can be increased by replacing the weak RBS 300 with the strong RBS 1200.

FIG. 3B includes fluorescence microphotographs of cells sequentially expressing all components of the genetic circuit in FIG. 3A (top panels) and quantitative flow cytometry analysis of the same samples (lower panels). FIG. 3C illustrates a time-lapse of the cells from FIG. 3B between the 2 hour and 4 hour mark following the 1.5 hour pulse of YhjH-mChy-PopZ expression induced with 0.02 mM IPTG. The same cells were also analyzed in conditions where either YhjH-mChy-PopZ or MrkH were not induced over the same time course, shown in FIGS. 3D and 3E. In FIG. 3D, MrkH was not expressed. In FIG. 3E, YhjH-mChy-PopZ was not expressed. Fluorescence microscopy and flow cytometry were performed using the methods described above.

FIGS. 3F-3H demonstrate quantitative flow cytometry analysis of cells transformed with pAC-YmP-B or pAC-YmP-S and pB-Mrk-GFP or pB-Mrk-rbsGFP plasmids, induced to express components of the genetic circuit in the same ways as described above. As observed in the strains bearing the split-GFP reporter, the ci-di-GMP signal was higher in the presence of the more active DGC Slr1143 (FIG. 3G), as compared to BphS (FIG. 3F). GFP signals in cells bearing the plasmid pB-Mrk-rbsGFP (FIG. 3F) were consistently higher than in cells bearing the pB-Mrk-GFP plasmid (FIG. 3H), corresponding with a higher GFP translation rate.

FIG. 3I illustrates a data plot of the average GFP intensity of YhJh-mChy-PopZ negative cells at the 4 hour post YhjH-mChy-PopZ induction course time point in strains containing the different circuit components described in relation to FIGS. 3A-3H. Compared to the circuits using the weaker RBS300 to drive mrkH expression, the enhanced circuit with RBS1200 (SEQ ID No. 19) increased GFP signal in cells lacking YhjH-mChy-PopZ by about six-fold.

FIGS. 3J and 3K illustrate characterization data of the YhjH-mChy-PopZ biochemical platform. In FIG. 3J, cells were transformed with the plasmid pAC-YmP-S and analyzed by fluorescence microscopy following a 90 minute pulse of YhjH-mChy-PopZ expression. The number of YhjH-mChy-PopZ foci per cell were counted at the end of the induction period and at the end of every hour during a 4 hour time course. Following induction, an average of 59% of the cells had two or more foci, often localized near opposite cell poles. As the cells divided over time, the fraction of YhjH-mChy-PopZ positive cells with only one polar focus increased from about 40% to greater than 90%. In FIG. 3K, flow cytometry data was utilized to assess the stability of the YhjH-mChy-PopZ foci between 2 hours and 4 hours post-induction. Fluorescence intensity of mChy remained constant for multiple rounds of cell division as the fraction of mChy-positive cells declined.

The YhjH-mChy-PopZ biochemical platform was further tested to determine whether it could be used to control production of a bioproduct requiring a multi-gene biosynthetic pathway. As illustrated in the genetic circuits of FIG. 3A, the coding sequences for acyl-CoA reductase (Ac-CoA red) (SEQ ID No. 1) from the Jojoba plant and wax ester synthase (WE-synthase) (SEQ ID No. 24) from Acinetobacter baylii were inserted downstream of the transcriptional reporter GFP. The combined activity of these enzymes produces long-chain neutral lipids, such as those naturally found in jojoba oil and spermaceti.

To clone WE-synthase and Ac-CoA reductase, the WE-synthase and Ac-CoA reductase coding sequences were first cloned as a poly-cistronic message (SEQ ID No. 30) under an araBAD promoter. The gene encoding Ac-CoA reductase was codon-optimized for expression in E. coli and chemically synthesized. WE-synthase was PCR-amplified from A. baylui. Both genes were cloned into pBAD-vectors via isothermal Gibson assembly, then amplified and cloned downstream of gfp in a pCDF:pMrkA-GFP(−) plasmid (plasmid map shown in FIG. 5N). The entire sequence of mrkAp-GFP-ac-CoA reductase-WE-synthase was then amplified and cloned into a pBAD-MrkH plasmid via isothermal assembly, resulting in the plasmid pBAD-M-G-W (plasmid maps shown in FIGS. 5G and 5J, respectively).

Utilizing the multi-gene biosynthetic circuits described above and illustrated in FIG. 3A, asymmetric cell division was induced and generated daughter cells that either contained the YhjH-mChy-PopZ biochemical platform or produced neutral lipids. Thus, it is contemplated that the YhjH-mChy-PopZ biochemical platform can be utilized to generate two distinct cells within an isogenic culture having different biosynthetic pathways. In other words, cells having the YhjH-mChy-PopZ biochemical platform were non-productive, while cells lacking the biochemical platform were productive. FIG. 3L illustrates expression data of cells transformed with the pBAD-M-G-W and pAC-YmP-S plasmids, 4 hours after a pulse of YhjH-mChy-PopZ expression with MrkH induction and BODIPY staining of the lipids as variables. Samples were fixed in 4% PFA for 30 minutes, then washed in PBS and stained with the lipophilic dye BODIPY 493/503²⁸ (488 nm) for 10 minutes. Fluorescence microscopy was performed using the methods described above, with mChy, and BODIPY (observed in Yellow (YFP) channel) signals overlaid on a phase contrast image (grayscale).

Optogenetic Control of Asymmetric Cell Division and Cell Differentiation

It is contemplated that optogenetic circuits, wherein external light exposure regulates expression of genes, may further be utilized in combination with or as an alternative to the small molecule-regulated circuits described above to control asymmetric cell division and cell differentiation. Several components of a photo-controllable transcriptional regulation system were thus incorporated into a genetic circuit to determine the feasibility of such an optogenetic circuit. These components include a light-activated histidine kinase CcaS, two additional genes hot and pcyA for synthesizing a phycocyanobilin chromophore, a cognate response regulator CcaR, and a CcaR-dependent promoter ccaRp. Upon exposure to green (535 nm) light, CcaS phosphorylates and activates CcaR, which then upregulates expression of the ccaRp promoter, located upstream of the YhjH-mChy-PopZ and thus linking the photo-controllable transcription regulation system to the production of the YhjH-mChy-PopZ biochemical platform. Furthermore, exposure to red (670 nm) light inactivates activity of the CcaS kinase.

FIG. 4A illustrates an optogenetic circuit controllable for PopZ expression. To make the optogenetic circuit in FIG. 4A, plasmids pSR58-6 and pNO286-3 (plasmid maps shown in FIGS. 5P and 5Q, respectively) with components for light-dependent gene expression were utilized as a backbone for further cloning. The pSR58-6 plasmid was PCR linearized, excluding the gfp coding sequence, and a PCR-amplified yhjH-mChy-popZ coding sequence was inserted in place of gfp, under the PcpcG2-172 promoter (later called ccaRp), to form a pCRP plasmid (plasmid map shown in FIGS. 5K and 5M). Then, the whole message encoding araBAD-p-mrkH-mrkAp-GFP was amplified from a pb-Mrk-rbsGFP plasmid and inserted into the PCR-linearized pCRP plasmid between the CamR (SEQ ID No. 5) and CCaR genes, thus resulting in the pCRPMG plasmid (plasmid map shown in FIG. 5K; coding sequence for YhjH-mChy-PopZ platform under control of the two-component light-activated transcription activation system (CcaS/CcaR) shown in SEQ ID No. 31). The pCSTS plasmid (plasmid map shown in FIG. 5L) was formed by inserting a tet-p-slr sequence (SEQ ID Nos. 20, 23) into the pNO286-3 plasmid between the pcyA and specR genes (SEQ ID No. 21) via isothermal Gibson assembly.

To determine whether asymmetric cell division and cell differentiation could be controlled by the above optogenetic circuit, MG-1655 DE3 cells were transformed with the pCRPMG and pCSTS plasmids and grown overnight at 37° C. with illumination by 650 nm red light to inactivate CcaS kinase activity. The overnight cells were then diluted 100 times and grown in 2 ml volumes in glass tubes with vigorous shaking and illumination with alternating red and green light. The cells were exposed to 1-hour cycles of 45 minutes with 650 nm red light and 15 minutes of 535 nm green light for a total of 3 hours to induce expression of the YhjH-mChy-PopZ biochemical platform. The cells were then diluted 20 times and released in 4 ml volumes in culture tubes and incubated under red light for 2 hours with shaking. The cells were subsequently diluted 10 times to maintain growth in early log phase after first 2 hour incubation in red light.

To re-induce expression and complete the cycle of YhjH-mChy-PopZ induction, the cells were again diluted 20 times and incubated for 3 hours under alternating red and green light with shaking, as described above. The complete cycle of YhjH-mChy-PopZ induction and chase was repeated 3 times with periodic dilution, as described above, to maintain log phase growth. Appropriate antibiotics were utilized in the growth media for all stages. To express MrkH and GFP, 0.2% L-arabinose was added during the chase periods.

FIGS. 4B and 4C illustrate changes in the fraction of YhjH-mChy-PopZ positive cells over multiple cycles of light stimulation. FIG. 4C further depicts data from two trials of the experiment. After the four hours of incubation in red light, the fraction of YhjH-mChy-PopZ positive cells was reduced to <10%, indicating that the cells divided and produced daughter cells lacking YhjH-mChy-PopZ. During the re-induction period of alternating red and green light, it was observed that the population of YhjH-mChy-PopZ cells was restored during periods of green light exposure, and returned to about 10% during subsequent exposure to constant red light. As depicted in the last panel of FIG. 4B, when MrkH was induced by L-arabinose in this system, cells lacking YhjH-mChy-PopZ expressed the GFP reporter. Thus, it is contemplated that light can be used to modulate the ratio of cell types in the cell population for prolonged periods of time and over repeated light exposure cycles.

To demonstrate that the optogenetic circuit described above can be utilized to differentiate cells on the basis of a physical trait, coding sequences motA-motB (SEQ ID Nos. 26, 27) for MotA-MotB flagellar stator proteins, involved in powering flagellar motors were inserted in the pCRPMG plasmid downstream of gfp to form the plasmid pCRPMG-Mot (plasmid map shown in FIG. 5M) having the genetic circuit of FIG. 4A. The pCRPMG and pCSTS plasmids were then transformed into a non-motile E. coli strain MG-1655 DE3 AmotA-motB. To form the non-motile strain, Lambda Red recombination was used to replace part of the motA-motB coding sequence with a kanR coding sequence of the pET28a plasmid (plasmid map shown in FIG. 5O).

FIG. 4D illustrates the effects of green light exposure on cell motility of MG-1655 DE3 ΔmotA-motB cells having the motA-motB-modified optogenetic circuit described above. The MG-1655 DE3 ΔmotA-motB cells were exposed to green light (left panel) or red light (right panel) for a time course of 7 hours. Continuous exposure to red light activated mrkA expression and rescued the motility defect of the cells in soft agar. Typically, high levels of c-di-GMP normally inhibit motility. However, high levels of MotA-MotB expression induced by red light overcame this effect (overexpression of MotA was previously shown to overcome inhibiting effect of c-di-GMP on motility). As depicted in FIGS. 4E, cells were further observed by fluorescence microscopy and tracked in liquid suspension to determine whether non-motile YhjH-mChy-PopZ cells could be induced to divide asymmetrically and produce motile daughter cells. Nearly all of the cells exposed to pulsed green light contained YhjH-mChy-PopZ foci, and these cells were non-motile. By contrast, the cells that were induced to divide asymmetrically by exposure to continuous red light consisted mostly of cells that did not have YhjH-mChy-PopZ foci and instead expressed GFP and were highly motile. Thus, it is contemplated that the optogenetic circuit described above may be utilized to control gene expression and physical cell traits, such as motility, through light-regulated patterning of asymmetric cell division and cell differentiation.

In summation, embodiments of the present disclosure provide synthetic genetic circuits for facilitating asymmetric cell division and cell differentiation in microbial cultures. The novel set of genetic components described herein utilize self-assembling macromolecular complexes as geometric cues to control cell behavior and generate complex microbial communities with two or more programmable cell types. The ability to facilitate multiple cell types can further be used to express one or more desired biosynthetic pathways among the cell types, enabling division of labor and spreading the fitness costs associated with target molecule production. Still further, by enabling physiochemical control of microbial populations, the ratio of cell types within a population may be dynamically adjusted. Thus, the overall productivity of microbial cultures used in bioproduct synthesis may be increased by controlling multiple facets of the biosynthetic pathways.

While the foregoing is directed to embodiments of the present disclosure, other and further embodiments of the disclosure may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow. 

What is claimed is:
 1. A method of controlling microbial cells, comprising: modifying microbial cells with a genetic circuit, the genetic circuit configured to produce: a localization factor exhibiting an asymmetric localization pattern as a basis for asymmetric cell division, the asymmetric cell division facilitating establishment of distinct cell types within a population of microbial cells; and a signaling factor linked to the localization factor to form a biochemical platform, the biochemical platform eliciting differentiable cell behavior in microbial cells that inherit the biochemical platform.
 2. The method of claim 1, wherein the localization factor is a protein that self-assembles into a macromolecular structure.
 3. The method of claim 1, wherein the localization factor is a polar organizing protein.
 4. The method of claim 3, wherein the polar organizing protein is PopZ or a homolog thereof.
 5. The method of claim 1, wherein the signaling factor is a protein that regulates gene expression in the microbial cells.
 6. The method of claim 1, wherein the signaling factor is a kinase.
 7. The method of claim 1, wherein the signaling factor is a phosphatase.
 8. The method of claim 1, wherein the signaling factor is a protease or protease adaptor protein.
 9. The method of claim 1, wherein the signaling factor is a member of a split-protein system.
 10. The method of claim 1, wherein the signaling factor is an enzyme that catalyzes production of a secondary messenger molecule.
 11. The method of claim 10, wherein the secondary messenger is a nucleotide-based secondary messenger.
 12. The method of claim 11, wherein the nucleotide-based secondary messenger is selected from the group consisting of c-di-GMP, cAMP, c-di-AMP, cGMP and c-di-AMP/GMP.
 13. The method of claim 10, wherein the genetic circuit is further configured to produce a transcriptional factor regulated by the secondary messenger.
 14. The method of claim 13, wherein the transcriptional factor is a transcriptional activator.
 15. The method of claim 1, further comprising: controlling expression of the biochemical platform by utilizing a physiochemical stimulus.
 16. The method of claim 15, wherein the physiochemical stimulus is a chemical inducer.
 17. The method of claim 15, wherein the physiochemical stimulus is light having a wavelength in a visible, infrared, or ultraviolet portion of the electromagnetic spectrum.
 18. The method of claim 17, wherein the physiochemical stimulus is light that controls activity of a histidine kinase.
 19. The method of claim 1, wherein microbial cells expressing the biochemical platform are biosynthetically non-productive.
 20. The method of claim 19, wherein asymmetric cell division is used to induce production in microbial cells that lack the biochemical platform.
 21. The method of claim 1, wherein the genetic circuit further comprises: a genetic element to induce expression of one or more enzymes involved in synthesis of a biosynthetic product.
 22. The method of claim 1, wherein the genetic circuit is used to separate a multi-step biosynthetic pathway into multiple stages, each stage being activated in a distinct differentiated cell type. 